The present invention relates generally to methods of treating chlamydial infections by administration of bactericidal/permeability-increasing (BPI) protein products.
BPI is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs or neutrophils), which are blood cells essential in the defense against invading microorganisms. Human BPI protein has been isolated from PMNs by acid extraction combined with either ion exchange chromatography Elsbach, J. Biol. Chem., 254:11000 (1979)! or E. coli affinity chromatography Weiss, et al., Blood, 69:652 (1987)!. BPI obtained in such a manner is referred to herein as natural BPI and has been shown to have potent bactericidal activity against a broad spectrum of gram-negative bacteria. The molecular weight of human BPI is approximately 55,000 daltons (55 kD). The amino acid sequence of the entire human BPI protein and the nucleic acid sequence of DNA encoding the protein have been reported in FIG. 1 of Gray et al., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference. The Gray et al. amino acid sequence is set out in SEQ ID NO: 1 hereto.
BPI is a strongly cationic protein. The N-terminal half of BPI accounts for the high net positive charge; the C-terminal half of the molecule has a net charge of -3. Elsbach and Weiss (1981), supra.! A proteolytic N-terminal fragment of BPI having a molecular weight of about 25 kD has an amphipathic character, containing alternating hydrophobic and hydrophilic regions. This N-terminal fragment of human BPI possesses the anti-bacterial efficacy of the naturally-derived 55 kD human BPI holoprotein. Ooi et al., J. Bio. Chem., 262:14891-14894 (1987)!. In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity against gram-negative organisms. Ooi et al., J. Exp. Med., 174:649 (1991).! An N-terminal BPI fragment of approximately 23 kD, referred to as "rBPI.sub.23," has been produced by recombinant means and also retains anti-bacterial activity against gram-negative organisms. Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992).
The bactericidal effect of BPI has been reported to be highly specific to gram-negative species, e.g., in Elsbach and Weiss, Inflammation: Basic Principles and Clinical Correlates, eds. Gallin et al., Chapter 30, Raven Press, Ltd. (1992). This reported target cell specificity was believed to be the result of the strong attraction of BPI for lipopolysaccharide (LPS), which is unique to the outer membrane (or envelope) of gram-negative organisms. Although BPI was commonly thought to be non-toxic for other microorganisms, including yeast, and for higher eukaryotic cells, it has recently been discovered that BPI protein products, as defined infra, exhibit activity against gram-positive bacteria, mycoplasma, mycobacteria, fungi, and protozoa. See allowed, co-owned, co-pending U.S. patent application Ser. No. 08/372,783 filed Jan. 13, 1995, the disclosures of which are incorporated herein by reference; co-owned, co-pending U.S. patent application Ser. No. 08/626,646, the disclosures of which are incorporated herein by reference; co-owned, co-pending U.S. patent application Ser. No. 08/372,105, the disclosures of which are incorporated herein by reference; and co-owned, co-pending U.S. patent application Ser. No. 08/273,470, the disclosures of which are incorporated herein by reference.! It has also been discovered that BPI protein products have the ability to enhance the activity of antibiotics against bacteria. See U.S. Pat. No. 5,523,288, the disclosures of which are incorporated herein by reference, and allowed, co-owned, co-pending U.S. patent application Ser. No. 08/372,783.!
The precise mechanism by which BPI kills gram-negative bacteria is not yet completely elucidated, but it is believed that BPI must first bind to the surface of the bacteria through electrostatic and hydrophobic interactions between the cationic BPI protein and negatively charged sites on LPS. LPS has been referred to as "endotoxin" because of the potent inflammatory response that it stimulates, i.e., the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to lipid A, reported to be the most toxic and most biologically active component of LPS.
In susceptible gram-negative bacteria, BPI binding is thought to disrupt LPS structure, leading to activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death. Elsbach and Weiss (1992), supra!. BPI is thought to act in two stages. The first is a sublethal stage that is characterized by immediate growth arrest, permeabilization of the outer membrane and selective activation of bacterial enzymes that hydrolyze phospholipids and peptidoglycans. Bacteria at this stage can be rescued by growth in serum albumin supplemented media Mannion et al., J. Clin. Invest., 85:853-860 (1990)!. The second stage, defined by growth inhibition that cannot be reversed by serum albumin, occurs after prolonged exposure of the bacteria to BPI and is characterized by extensive physiologic and structural changes, including apparent damage to the inner cytoplasmic membrane.
Initial binding of BPI to LPS leads to organizational changes that probably result from binding to the anionic groups of LPS, which normally stabilize the outer membrane through binding of Mg.sup.++ and Ca.sup.++. Attachment of BPI to the outer membrane of gram-negative bacteria produces rapid permeabilization of the outer membrane to hydrophobic agents such as actinomycin D. Binding of BPI and subsequent gram-negative bacterial killing depends, at least in part, upon the LPS polysaccharide chain length, with long O-chain bearing, "smooth" organisms being more resistant to BPI bactericidal effects than short O-chain bearing, "rough" organisms Weiss et al., J. Clin. Invest. 65: 619-628 (1980)!. This first stage of BPI action, permeabilization of the gram-negative outer envelope, is reversible upon dissociation of the BPI, a process requiring high concentrations of divalent cations and synthesis of new LPS Weiss et al., J. Immunol. 132:3109-3115 (1984)!. Loss of gram-negative bacterial viability, however, is not reversed by processes which restore the envelope integrity, suggesting that the bactericidal action is mediated by additional lesions induced in the target organism and which may be situated at the cytoplasmic membrane (Mannion et al., J. Clin. Invest. 86: 631-641 (1990)). Specific investigation of this possibility has shown that on a molar basis BPI is at least as inhibitory of cytoplasmic membrane vesicle function as polymyxin B (In't Veld et al., Infection and Immunity 56:1203-1208 (1988)) but the,exact mechanism as well as the relevance of such vesicles to studies of intact organisms has not yet been elucidated.
Chlamydia are nonmotile, gram-negative, obligate intracellular bacteria that have unusual biological properties which phylogenetically distinguish them from other families of bacteria. Chlamydiae are presently placed in their own order, the Chlamydiales, family Chlamydiaceae, with one genus, Chlamydia. Schachter and Stamm, Chlamydia, in Manual of Clinical Microbiology, pages 669-677, American Society for Microbiology, Washington, D.C. (1995).! There are four species, Chlamydia trachomatis, C. pneumoniae, C. psittaci and C. pecorum, which cause a wide spectrum of human diseases. In developing countries, C. trachomatis causes trachoma, the world's leading cause of preventable blindness. Over 150 million children have active trachoma, and over 6 million people are currently blind from this disease. In industrialized countries, C. trachomatis is the most prevalent sexually transmitted disease, causing urethritis, cervicitis, epididymitis, ectopic pregnancy and pelvic inflammatory disease. Last year alone, an estimated 300 million people contracted sexually transmitted chiamydial infections. Among the 250,000 cases of pelvic inflammatory disease per year in the United States, approximately 25,000 women are rendered infertile each year. Neonatal C. trachomatis infections, contracted at birth from infected mothers, cause hundreds of thousands of conjunctivitis cases per year, of which about half of these infected infants develop pneumonia. Recently, C. pneumoniae has been implicated as a common cause of epidemic human pneumonitis. Members of the genus are not only important human pathogens, but also cause significant morbidity in other mammals and birds. Thus, chlamydia are one of the most ubiquitous pathogens in the animal kingdom. Zhang et al., Cell, 69:861-869 (1992).!
Their unique developmental cycle differentiates them from all other microorganisms. They are obligate intracellular parasites that are unable to synthesize ATP, and thus depend on the host cells' energy to survive. Unlike viruses, they always contain both DNA and RNA, divide by binary fission, contain ribosomes, and can synthesize proteins. Chlamydia have cell walls similar in structure to those of gram-negative bacteria, and all members of the genus carry a unique LPS-like antigen, termed complement fixation (CF) antigen, that may be analogous to the LPS of certain gram-negative bacteria. Schachter and Stamm, supra.! Chlamydia also carry a major outer membrane protein (MOMP) that contains both species and subspecies-specific antigens.
The infectious form of chlamydia is the elementary body (EB), which infects mammalian cells by attaching to the host cell and entering in a host-derived phagocytic vesicle (endosome), within which the entire growth cycle is completed. The target host cell in vivo is typically the columnar epithelial cell, and the primary mode of entry is believed to be recept-ormediated endocytosis. Once the EB has entered the cell, it reorganizes into a reticulate body (RB) that is larger than the EB and metabolically active, synthesizing DNA, RNA and proteins. The EBs are specifically adapted for extracellular survival, while the metabolically active RBs do not survive well outside the host cell and seems adapted for an intracellular milieu. After approximately 8 hours, the RBs begin dividing by binary fission. As they replicate within the endosomes of host cells, they form characteristic intracellular inclusions that can be seen by light microscopy. After a period of growth and division, the RBs reorganize and condense to form infectious EBs. The developmental cycle is complete when host cell lysis or exocytosis of chlamydia occurs, releasing the EBs to initiate another cycle of infection. The length of the complete developmental cycle, as studied in cell culture models, is 48 to 72 hours and varies as a function of the infecting strain, host cell and environmental conditions. Beatty et al., Microbiol. Rev., 58(4):686-699 (1994).!
It has been demonstrated, at least for C. trachomatis, that attachment of the chlamydia organism to host cells is mediated by a heparan sulfate-like glycosaminoglycan (GAG) present on the surface of the chlamydia. Treatment of chlamydia with either purified heparin, heparin sulfate, or heparin receptor analogs (such as platelet factor 4 and fibronectin, both of which are known to bind heparin sulfate), inhibited the attachment and infectivity of chlamydia to host cells. Inhibition was not seen with non-heparin GAGs, such as hyaluronate, chondroitin sulfate, or keratin sulfate. Treatment of C. trachomatis with heparitinase reduced attachment and infectivity by greater than 90%; subsequent treatment with exogenous heparan sulfate was able to restore the ability of treated organisms to attach to host cells in a dose-dependent manner. Other GAGs such as hyaluronate, chondroitin sulfate, or keratin sulfate did not restore attachment ability. These data suggest that a heparin sulfate-like GAG mediates attachment of chlamydia to host cells by bridging mutual GAG receptors on the host cell surface and on the chlamydial outer membrane surface. Zhang et al., Cell, 69:861-869 (1992).!
C. trachomatis is almost exclusively a human pathogen and is responsible for trachoma, inclusion conjunctivitis, lymphogranuloma venereum (LGV), and genital tract diseases. Schachter and Stamm, supra.! Within this species, serotypes A, B, Ba, and C have been associated with endemic trachoma, the most common preventable form of blindness in the world. Trachoma is a chronic inflammation of the conjunctiva and the cornea, which is not sexually transmitted. The potentially blinding sequelae of trachoma include lid distortion, trichiasis (misdirection of lashes), and entropion (inward deformation of the lid margin). These can cause corneal ulceration followed by loss of vision. Serotypes L1, L2, and L3 of C. trachomatis are associated with LGV. Untreated, lymphogranuloma venereum progresses through three stages, each more severe than the preceding one. The primary lesion, if present, appears on the genitals. The second stage is a bubonic state marked by regional lymphadenopathy, during which the buboes may suppurate and develop draining fistulas. Rectal strictures and lymphatic obstruction can appear in the tertiary stage. Lymphogranuloma venereum is a common problem in developing countries with tropical or subtropical climates, especially among the lower socioeconomic groups.
C. trachomatis is also the most common agent of sexually transmitted disease. In men, serotypes D through K are the major identifiable causes of nongonococcal urethritis, and also cause epididymitis, Reiter's syndrome, and proctitis. Chlamydial infections are not easily identified in men by clinical symptoms alone, because the infection may be asymptomatic and because other pathogens cause similar symptoms. Chlamydial urethritis occurs twice as frequently as gonococcal urethritis (gonorrhea) in some populations, and its incidence is on the increase. Even when N. gonorrhea is shown to be present, the urethritis may be due to a dual or multiple infection involving a second organism. Concurrent C. trachomatis and N. gonorrhoea infections have been reported in about 25 percent of men with gonorrhea. Epididymitis is the most important complication of chlamydial urethritis in men. C. trachomatis causes one of every two cases of epididymitis in younger men in the United States, with sterility a possible result. Reiter's syndrome is another manifestation of chlamydial infection in men. It is a painful systemic illness that classically includes symptoms of urethritis, conjunctivitis and arthritis. Urethritis and arthritis are by far the most frequent combination; it appears that the chlamydial urethral infection may trigger the arthritis. C. trachomatis can also cause proctitis (anal inflammation), particularly in homosexual men.
In women, chlamydial infecton with the sexually transmitted serotypes results in cervicitis, urethritis, endometritis, salpingitis, and proctitis; serious sequelae of salpingitis include tubal scarring, infertility, and ectopic pregnancy. Unrecognized chlamydial infections in women are common. Approximately 50 percent of women infected with chlamydia are asymptomatic. C. trachomatis causes mucopurulent cervicitis and the urethral syndrome, as well as endometritis and salpingitis. These upper genital tract chlamydial infections may cause sterility or predispose to ectopic pregnancies and are the gravest complications of chlamydial infections in women. Ten percent of all maternal deaths are due to ectopic pregnancies. C. trachomatis causes over 30 percent of the cases of mucopurulent cervicitis. As many as one-half of the women with gonococcal cervicitis have a concomitant chlamydial infection. If the gonococcal infection is treated with penicillin, the concomitant chlamydial cervicitis will continue undetected and untreated, and may progress to pelvic inflammatory disease (salpingitis), which can lead to sterility and ectopic pregnancies. C. trachomatis is a cause of the urethral syndrome in women. Chlamydial infections may ascend from the cervix to the endometrium, where C. trachomatis has been found in the epithelial lining of the uterine cavity. It is estimated that about one-half of all women will cervicitis have endometritis. Salpingitis. a major cause of ectopic pregnancies and infertility, is the most serious complication of female genital infections. Upper abdominal pain is the predominant symptom of perihepatitis. Both C. trachonatis and N. gonorrhoea can cause perihepatitis. This condition occurs almost exclusively in women in whom the infecting organisms spread to the surface of the liver from inflamed fallopian tubes.
Women infected with C. trachomatis may also pass the disease to their newborn as it passes through the infected birth canal. These newborns most often develop inclusion conjunctivitis or chlamydial pneumonia, but may also develop vaginal, pharyngeal, or enteric infections. Though not blinding, inclusion conjunctivitis can become chronic, causing mild scarring and pannus formulation if left untreated. During passage through the birth canal, up to two-thirds of babies born to mothers with chlamydial genital infections will also become infected. With as many as one in ten pregnant women having chlamydial cervicitis in some parts of the world, the risk to newborns is considerable. Chlamydial pneumonia occurs in 10 percent to 20 percent of infants born to infected mothers. C. trachomatis is responsible for 20 percent to 60 percent of all pneumonias during the first 6 months of life.
C. trachomatis strains are sensitive to the action of tetracyclines, macrolides and sulfonamides and produce a glycogen-like material within the inclusion vacuole that stains with iodine.
C. psittaci strains infect many avian species and mammals, producing such diseases as psittacosis, ornithosis, feline pneumonitis, and bovine abortion. Schachter and Stamm, supra.! C. psittaci is ubiquitous among avian species, and infection in birds usually involves the intestinal tract. The organism is shed in the feces, contaminates the environment, and is spread by aerosol. C. psittaci is also common in domestic mammals. In some parts of the world, these infections have important economic consequences, as C. psittaci is a cause of a number of systemic and debilitating diseases in domestic mammals and, most important, can cause abortions. Human chlamydial infections from this agent usually result from exposure to an infected avian species, but may also occur after exposure to infected domestic mammals. This species is resistant to the action of sulfonamides and produces inclusions that do not stain with iodine.
C. pneumoniae has less than 10% DNA relatedness to the other species and has pear-shaped rather than round elementary bodies (EBs). Like C. trachomatis, it appears to be exclusively a human pathogen without an animal reservoir. C. pneumoniae has been identified as the cause of a variety of respiratory tract diseases and is distributed worldwide. Schachter and Stamm, supra.! Infections appear to be commonly acquired in later childhood, adolescence, and early adulthood, resulting in seroprevalences of 40 to 50% in 30 to 40-year-old people. Manifestations of infection include pharyngitis, bronchitis, and mild pneumonia, and transmission is primarily via respiratory secretions. In seroepidemiological studies, these infections have been linked with coronary artery disease, and their role in atherosclerosis is currently under intense scrutiny.
The role of C. pecorum as a pathogen is not clear, and specialized reagents are required for its identification.
The recommended procedure for primary isolation of chlamydia is cell culture. Chlamydia will grow in the yolk sac of the embryonated hen egg, as well as in cell culture (with some variability). C. trachomatis can infect several cell lines, such as McCoy's heteroploid murine cells, HeLa 229 cells, BHK-21 cells, or L-929 cells. HL cells and Hep-2 cells may be more sensitive for the recovery of C. pneumoniae. The most common technique involves inoculation of clinical specimens into cycloheximide-treated McCoy cells. The basic principle involves centrifugation of the inoculum onto the cell monolayer, incubation of the monolayers for 48 to 72 hours, and demonstration of typical intracytoplasmic inclusions by appropriate immunofluorescence, iodine or Giemsa staining procedures. Cell culture generally requires two to six days to complete because of the incubation time required.
Chlamydia may also be detected in samples by the direct fluorescent antibody (DFA) test, in which slides are incubated with fluorescein-conjugated monoclonal antibodies, and fluorescing elementary bodies are detected using a fluorescent microscope. This test has approximately 80% to 90% sensitivity and 98% to 99% specificity compared with cell cultures when both tests are performed under ideal circumstances. Schachter and Stamm, supra.!
A number of commercially available products can detect chlamydial antigens in clinical specimens by using enzyme immunoassay (EIA) procedures. Most of these products detect chlamydial LPS, which is more soluble than MOMP. Without confirmation, the tests have a specificity on the order of 97%. Schachter and Stamm, supra.! Several nucleic acid probes are also commercially available. One commercially available probe test (GenProbe) utilizes DNA-RNA hybridization in an effort to increase sensitivity by detecting chlamydial RNA.
The complement fixation (CF) test is the most frequently performed serological test, and measures serum level of complement-fixing antibody (antibody to the group CF antigen). It is useful for diagnosing psittacosis, in which paired acute- and convalescent-phase sera often show four-fold or greater increases in titer. The same seems to be true for many C. pneumoniae infections. Approximately 50% of these infections are CF-positive, although it may take 24 weeks to detect seroconversion. CF testing may also be useful in diagnosing LGV, in which single-point titers greater than 1:64 are highly supportive of this clinical diagnosis. Schachter and Stamm, supra.! High titers of complement-fing antibodies are not found in chlamydial conjunctivitis or genital tract infections, and therefore are not sensitive for these infections.
The microimmunofluorescence (micro-IF) method is a much more sensitive procedure for measuring anti-chlamydial antibodies. This indirect fluorescent antibody technique uses antigens prepared by infecting the yolk sacs of fertile chick embryos with each chlamydial serotype. Serial dilutions of patient serum are added to the prepared antigens, and the level of antibody in the blood sample is determined with the use of immunofluorescence. Trachoma, inclusion conjunctivitis, and genital tract infections may be diagnosed by the micro-IF technique if appropriately timed paired sera can be obtained, but the procedure is of limited clinical utility because diagnosis requires demonstration of a four-fold or greater change in antibody titer in paired specimens. and because patients with superficial genital infections such as urethritis may not have a change in titer. However, a high antibody titer in a single serum specimen from a patient with Reiter's syndrome and a high IgM titer in the serum of an infant with pneumonia are helpful in establishing a diagnosis.
Strain-to-strain variation in antimicrobial susceptibility profiles and newly acquired drug resistance are both very infrequent among chlamydia. Among the drugs most active in vitro against C. trachomatis, C. pneumoniae, and C. psittaci are the tetracyclines, such as tetracycline and doxycycline, the macrolides, such as erythromycin and azithromycin, the quinolones, such as ciprofloxacin and ofloxacin, chloramphenicol, rifampin, clindamycin and the sulfonamides. The tetracyclines and macrolides have generally been the mainstays of therapy for infections due to chlamydia. Schachter and Stamm, supra; Goodman and Gilman, The Pharmacological Basis of Therapeutics, 9th ed., McGraw-Hill, New York, N.Y. (1996).!
Antimicrobial susceptibility testing is infrequently performed for chlamydial infections, but may be conducted as follows. The organisms for testing are grown for at least two passages in cells cultured in antibiotic-free media before being harvested. An adjusted inoculum of .about.100 inclusion-forming units per microtiter well is then used to infect antibiotic-free cell monolayers. After centrifugation of the inoculum onto the monolayer, serial dilutions of the test antibiotic can be added either immediately or at various time intervals over the next 24 hours. After 48 hours, fluorescein-conjugated monoclonal antibodies are use to identify minimum inhibitory concentration (MIC), i.e., the highest antibiotic dilution that inhibits intracellular inclusion formation. Generally, monolayers are also disrupted and further passaged to define the minimum bactericidal concentration (MBC), i.e., the highest antibiotic dilution that prevents viable chlamydia from being detected in passage (MBC).